1. Field of the Invention
The present invention relates to a biochip for detecting phosphorylation and a method for detecting phosphorylation using the same, more precisely a biochip prepared by integrating a fusion protein produced from the recombination of a substrate of a kinase selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase), and the elevated protein such as Selenomonas ruminantium membrane protein on a matrix surface coated with an active group, a kit for detecting phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope and a method for detecting phosphorylation using the same.
2. Description of the Related Art
Recent biotechnology industry is characterized by the fusion of biology with related technologies in the fields of electronics, computer science and mechanical engineering. Biological studies require a new system to approach an individual biological subject as a whole and also require a novel approaching method performing experiments in a large scale with a small amount of samples. Accordingly, a biochip is expected to play a crucial role in the field of bioinformation analysis using genes, proteins and cells and in use of the bioinformation.
According to the completion of human genome project, studies on micro-analysis system such as DNA chip or protein chip for the analysis of genes or proteins are actively undergoing in the overall biological industry. Biochip market is expected to be growing greatly and has been a major target of development in bio-industry in Korea.
Protein chip or peptide chip is a kind of automatic analysis device for simultaneous analysis of tens or thousands of proteins or peptides fixed on a small matrix, which is the next generation biosensor different from DNA chip in analysis mechanism and applying field. Protein chip is a key technique to disclose functions of biomolecules interacting specifically to specific proteins and to develop a novel method for preventing and treating disease, particularly those diseases untreatable so far by the conventional arts, based on functional analysis and network analysis of protein.
Protein chip techniques are largely divided into three categories; protein microarray techniques related to chip production, analysis techniques for quantitatively measuring and comparing interactions between proteins by observing proteins fixed on array and application techniques of protein chip. The first core technique, protein microarray chip is prepared by different methods according to techniques to analyze protein chip. For example, in the case of using SPR (Surface Plasmon Resonance), proteins have to be fixed on a thin metal film such as a thin gold film. Therefore, a thin gold film construction method and a protein fixation technique have to be developed together. In the case of using a fluorescent material, proteins are fixed directly on a slide glass for analysis. So, the proteins have to be labeled with fluorescent materials.
The second core technique, the analysis technique of protein chip includes SPR, mass spectrometry, fluorometry, electrochemical analysis method and nano-imaging technique such as Ellipsometry, which has been developed very competitively so far. Fluorometry based on DNA chip analysis has been most widely used so far, but each method has been recognized to have merits and demerits, and which protein chip analysis method is most appropriate for disease is still undecided.
The last core technique, the application technique of protein chip has the biggest potential for the development and still leaves a lot to discover.
The newly developed techniques related to protein chip are classified into four categories as follows.
(1) A technique to analyze interaction between DNA and protein on the chip by using DNA microarray. On the chip, single-stranded oligonucleotide is converted into double-stranded oligonucleotide, to which specific DNA sequence restriction enzyme is reacted. Then, DNA-protein interaction was examined by measuring digestion. So, this technique is effective in identifying a novel DNA binding protein and in disclosing the characteristics thereof (Bulyk, M. L. et al., Nature. Biotechnol., 17:573-577, 1999).
(2) A technique to analyze diverse enzymes including restriction enzyme, peroxidase, phosphatase and protein kinase, and antigen-antibody reaction on the chip (US Patent Publication No. 2002/0055186A1; WO 01/83827A1; Braunwalder A. et al., Anal. Biochem., 234:23-26, 1996; Houseman B. et al., Nature Biotechnol., 20:270-274, 2002; Ruud M. et al., Nature Biotechnol., 18:989-994, 2000). In particular, this technique can be applied in mass-analysis, biochemical analysis, new drug candidate analysis and disease diagnosis by examining protein-protein interaction, kinase-peptide substrate reaction and protein-ligand binding reaction. However, when a kinase specific substrate peptide or a low-molecular weight protein is fixed, bovine serum albumin (BSA) is necessarily added in order to prevent non-specific fixation, which buries the protein fixed thereon. Besides, when different antibodies are fixed on the chip to be reacted with fluorescein labeled antigen mixture, only 60% of the antibodies were quantified and only 23% of the antibodies were qualified (MacBeath G. et al., Science, 289:1760-1763, 2000; Haab B. et al., Genome Biol. 2: research 0004, 2001).
(3) A technique to analyze massive proteins expressed on the chip from cDNA library (WO 01/83827, WO 02/50260). This technique is effective in large scale measurement of biochemical activity of a protein (Heng Zhu, et al., Nature genetics, 26:283-289, 2000).
(4) A method to analyze a sample by using the technique of regulating orientation of biomolecules at molecular level using affinity tag and forming a stable and single layer of biomolecules on the surface of the chip (US Patent Publication No. 2002/0055125A1; U.S. Pat. No. 6,406,921; Paul J. et al., JACS, 122:7849-7850, 2000; RaVi A. et al., Anal. Chem., 73:471-480, 2001; Benjamin T. et al., Tibtech., 20:279-281, 2002). For example, a protein is expressed as a His-tag fusion protein, which is reacted to a chip fixed with Ni-NTA functional group, leading to fixation. The protein is then expressed as an intein fused protein or retains the activity, which not only makes the purification easy but also makes the protein on the chip more stable and active by fixing in a regular direction on the avidin treated chip (Zhu et al., Science, 293:2101-2105, 2001; Marie-Laure L. et al., JACS 124:8768-8769, 2002). And, the protein is expressed on the chip as a fusion protein using a supporter specific protein (calmodulin, etc) and tag (poly cysteine, lysine, histidine, etc) and fixed. The protein is purified by using protein-protein interaction and further used for SPR (surface plasmon resonance) and FACS (fluorescence activated cell sorter) (Hentz et al., Anal. Chem., 68:3939-3944, 1996; Hodneland et al., PNAS, 99:5048-5052, 2002; Kukar et al., Anal. Biochem., 306:50-54, 2002; U.S. Pat. No. 6,117,976).
Kinase is a protein enzyme involved in signal transduction pathways to induce a series of reactions in vivo and hence it is considered to be an effective target for a drug. Kinase provides γ-phosphorylated group to serine, threonine and tyrosine residues of the specific sequence of a target protein from ATP provided in cells, by which it is involved in signal transduction pathway in eukaryotic cells and various diseases (Hunter, T., Cell 100:113-127, 2000; Zhang, Z. Y., Curr. Opin. Chem. Biol. 5:416-423, 2001). The conventional method to study the activity of kinase is to examine cell membrane using a radio-isotope, but this method is very slow-going and requires a great labor. Another conventional method to measure kinase and its receptor in a large scale is ELISA (enzyme-linked immunosorbent assay) or a method using antibody. ELISA is a comparatively accurate method but takes a lot of time and requires a huge amount of samples. The method using an antibody facilitates mass-analysis but requires high costs and the processes are very complicated.
Promega Co., USA provides a phosphorylation assay kit using a membrane having high absorptiveness to ATP labeled with a radio-isotope and biotinated kinase substrate. However, this kit is limited in mass-analysis. This company also provides an analysis method without using a radio-isotope but using moving difference on electrophoresis caused by changes of net charge of the substrate after phosphorylation of kinase. But, this method requires high costs for mass-analysis. Except for the said four protein chip related techniques, there is no method available for screening the activity of protein kinase in a large scale by using protein chip or peptide chip. Therefore, it is urgently required to develop a new system facilitating accurate and fast analysis with low costs.
The present inventors constructed a biochip for the detection of phosphorylation in Korean Patent Application No. 2007-0070049, in which kinase substrate is integrated on the surface of a matrix directly or as a fusion protein fused to E. coli malic enzyme, but further studies on the biochip using PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) substrate are required.
Thus, the present inventors further studied and constructed a recombinant fusion protein from the fusion of PKC, cdc2-PK or DNA PK substrate with Selenomonas ruminantium membrane protein and then completed this invention by constructing a protein chip on which the recombinant fusion protein is integrated on a matrix surface coated with an active group and measuring phosphorylation by the kinase.